Abnormal large-scale chromosome structure is a hallmark of cancer and many other human diseases, but the mechanisms linking chromosome structure to function remain unresolved. The temporal order of replication is developmentally regulated at the level of large (400-800kb) replication domains that correspond to stable units of chromosome structure. Our long-term goal is to understand the role of largescale chromosome architecture in cell fate transitions. The objective of this application is to determine the causal interdependency of changes in replication timing and its correlated chromosome properties during human embryonic stem cell (hESC) differentiation. Our central hypothesis is that differentiation signals directiy modify replication timing to alter chromatin composition, which will in turn influence 3D folding in the next cell cycle, contributing to the robustness of transcription networks. Our rationale is that knowledge of causal relationships is an essential first step of mechanistic studies linking large-scale chromosome structure to cell fate transitions. Aimi will determine the order in which changes in replication timing, histone modifications, 3P chromatin interactions and transcription occur in response to differentiation and their dependence upon completion of prior events. Preliminary data describe newly developed hESC differentiation and cell cycle synchronization methods that can achieve this goal. Aim2 will test the hypothesis that human Rifl protein, which we recentiy identified as essential to maintain replication timing, is redistributed during differentiation to regulate replication timing. Gene disruption, genome wide ChlP, and single cell methods will localize Rifl and determine its role in regulating replication and transcription. These Aims are significant because identifying causal relationships and molecular players involved will remove a major obstacle in the field, paving the way to investigate mechanisms linking large-scale chromosome structure to cell fate commitment and, ultimately, human disease. The work is innovative in developing a system to study cell cycle regulated events in response to differentiation and in pioneering investigations into the newly identified role of Rifl in replication timing during early human development.